Bioconjugates, such as protein-oligonucleotide conjugates, have been employed in a wide variety of molecular biology applications. For example, bioconjugates are used in biochemical assays and diagnostic assays to improve assay sensitivity. Bioconjugates, such as oligonucleotides conjugated to antibodies or enzymes, have been used as hybridization probes in immunoassays or as probes in the development of sensitive nucleic acid-based diagnostic assays. Such conjugates may be prepared by a variety of methods, such as glutaraldehyde crosslinking, maleimide-thiol coupling, isothiocyanate-amine coupling, hydrazone coupling, oxime coupling and Schiff base formation/reduction.
Despite the promise that bioconjugates hold in the area of biomedical research, such as improving assay sensitivity, simplifying nucleic acid detection schemes, clinical studies, development of both in vitro and in vivo diagnostic assays as well as in vivo therapies and the like, bioconjugates have not yet achieved their full potential in these molecular biology, biomedical and diagnostic applications. This deficiency is due, in part, to the less than quantitative preparation of bioconjugates, which may involve multiple steps and may require, for example, the protein, the oligonucleotide, or both, to be modified with the appropriate linking moiety and then purified before being combined and reacted with each other. Often the modification reaction may have a lengthy reaction time and may result in forming an unstable protein or oligomer intermediate that must be purified and used immediately. For these and other reasons, the yields to prepare these bioconjugates are highly variable and are greatly dependent on what techniques are used.
Another reason that has hindered the widespread use of bioconjugates, is the methods used to purify and isolate bioconjugates. Because of the inefficiencies in the conjugation chemistries used to prepare bioconjugates, often the resulting bioconjugate product may require several purification steps to obtain a purified bioconjugate, which can have a detrimental effect on the stability or activity of the final bioconjugate, its yield as well as be time consuming and expensive to prepare and/or purify.
Up to this point, the purification of bioconjugates has been accomplished using, for example, size exclusion chromatography, or occasionally, ion exchange chromatography. The requirement for HPLC chromatography for purification of bioconjugates has been a significant barrier for the routine use of bioconjugates, such as antibody-oligonucleotide bioconjugates in diagnostic assays. For these and other reasons, the costs of preparing and purifying bioconjugates have been expensive and have been difficult to make with reproducible results.
Therefore, there remains a great need for methods that provide a more efficient, robust, mild, simple and high-yielding purification of such bioconjugates to provide high purity bioconjugates for use in biomedical research and diagnostic assays.
Developments in conjugation chemistry have improved the efficiency of preparing bioconjugates. For example, SoluLink™ has disclosed conjugation chemistry that can be used to prepare an antibody-oligonucleotide bioconjugate with at least 80% efficiency. Accordingly, the preparation of bioconjugates using efficient conjugation chemistries has allowed for the ability to explore efficient, mild, robust, simple and high yielding purification methods to provide bioconjugates, such as antibody-oligonucleotide bioconjugates, in high yield having high purity to facilitate their use in molecular biology, biomedical and diagnostic research and application.
The present disclosure provides methods, systems and/or kits for the preparation, purification and/or isolation of bioconjugates that have been prepared via efficient conjugation chemistry, wherein the bioconjugate comprises at least one biomolecule conjugated to another biomolecule, for example, at least one oligonucleotide conjugated to an antibody or protein.